Between 15 and 20% of couples have difficulty conceiving; failures of the reproductive system thus affect many individuals. In females, understanding the control of ovulation is critical for helping those with infertility conceive singe, as opposed to multiple, births, and for developing novel methods to prevent unwanted pregnancy in manners that are consistent with the acceptable social mores of most of the population, while minimizing side effects. The goal of this proposal is to increase our understanding of the generation of the central neural signal that ultimately leads to ovulation. This signal is provided by a shift in output of gonadotropin-releasing hormone (GnRH) neurons from one that is strictly episodic, producing on/off GnRH pulses that drive pituitary hormone release, to one in which GnRH release is continuously elevated for several hours. Estradiol initiates this GnRH surge, which induces the luteinizing hormone (LH) surge that subsequently triggers ovulation. To induce the GnRH surge, central estradiol action switches from negative feedback to positive feedback. Ovariectomized (OVX) mice treated with constant physiological levels of estradiol (OVX+E) undergo daily shifts from negative to positive feedback that are timed to the light-dark cycle, allowing mechanistic studies in a reduced variable model. In ovary-intact mice, this switch in estradiol feedback mode occurs on proestrus. Previous work in the daily surge model established several mechanisms engaged by estradiol that would lead to suppression of GnRH neurons during negative feedback and activation of these cells during positive feedback. In the proposed work, these findings will be extended with experiments that range from reductionist investigation of neurobiological mechanisms to whole animal studies, all aimed at elucidating the upstream neuronal networks engaged by estradiol to regulate GnRH neurons and surge generation. In Aim 1, we will study kisspeptin neurons in the anteroventral periventricular (AVPV) region, postulated to mediate estradiol positive feedback. We will determine how their inputs and intrinsic properties change with estradiol and time of day. We will also study how estradiol feedback alters functional connectivity between kisspeptin and GnRH neurons using paired recordings in brain slices. Preliminary data indicate firing pattern, intrinsic properties and neurotransmission to AVPV kisspeptin neurons are altered both by estradiol and/or time of day. In Aim 2, we will study the mechanisms by which an acute stress disrupts the LH surge. This aim will test the neurobiological mechanisms that are disrupted by stress, and determine effector cells using genetic and surgical approaches. This aim will also expand our knowledge of mechanisms underlying the surge to the natural cycle. Preliminary data indicate a diurnal pattern to stress inhibition of surge generation, that the stress peptide corticotropin- releasing hormone inhibits GnRH neurons and that this is exacerbated by gonadal factors. Integration of the data resulting from the study of an excitatory and an inhibitory afferet network into existing knowledge will increase our understanding of the central neuronal control of ovulation by estradiol.